Week 15
Enzyme assay and inhibition assays were done this week. Virtual was attempted, but due to fault in preparation of files, was not able to run on the processors and yield any results.
Figure 6: Scatterplot of the second inhibitor tried on YopH, showing absorbances at 410nm for samples A-I in order, where A had no enzyme, and all other samples had 40 uL of enzyme, and sample A and D contained 1.6 uL of compound, samples B and C recieved 1.6 uL of 50% DMSO, sample E recieved 3.2 uL of compound, sample F contained 4.8 uL of compound, sample G contained 6.4 uL of compound, sample H contained 9.6 uL of compound and sample I contained the positive control orthovanadate
Figure 5: Scatterplot of the first inhibitor tried on YopH, showing absorbances at 410nm for samples A-I in order, where A had no enzyme, and all other samples had 40 uL of enzyme, and sample A and D contained 1.6 uL of compound, samples B and C recieved 1.6 uL of 50% DMSO, sample E recieved 3.2 uL of compound, sample F contained 4.8 uL of compound, sample G contained 6.4 uL of compound, sample H contained 9.6 uL of compound and sample I contained the positive control orthovanadate
Figure 4: Spectrophotometer output from inhibition assay with a different compound, showing almost no inhibition at any of the volumes of the compound.
Figure 3: Spectrophotometer output from first inhibition assay, showing sligh inhibition, but no notable inhibition until highest volume of the inhibiting compound.
Figure 2: Excel graph of data points from the enzyme assay, showing a relatively linear relationship between the amount of enzyme, and absorbance at 410 nm.
Figure 1: Spectrophotometer output graph for the enzyme assay, showing a steady increase in the absorbance as the volume of enzyme increases.
Week 14
On this week, expression, purification, FPLC, characterization, and storage were completed.
Image 8: The combined samples from the concentration of elution one, after glycerol storage. This yield correlates to a concentration of 45.27 uM, which was high enough to use for assays.
Image 7: Nanodrop of Elution 1, sample 1 after third round of concentration by centrifugation. This yield was deemed high enough, so the sample was not concentrated again.
Image 6: Nanodrop of Elution 1, sample 1 after second round of concentration by centrifugation. This yield was not high enough, so the sample was concentrated again
Image 5: Nanodrop of Elution 1, sample 2 after FPLC and first round of concentration. This yield was high enough that it did not need to be concentrated further.
Image 4: Nanodrop of Elution 1, sample 1 after initial round of concentration by centrifugation. This yield was not high enough, so the sample was concentrated again.
Image 3: Nanodrop of Elution 1 immediately after FPLC. This yield was not hight enough, so the sample was concentrated further.
Image 2: Second PAGE gel of elutions from expression and purification before drying; they show some contamination, which necessitated FPLC
Image 1: First page gel of elutions from expression and purification before drying; they show some contamination, which necessitated FPLC.
Week 13
On this week, mini-prep and submitting to DNA sequencing were performed. The DNA sequencing results came back blank or with very, very few codons. My partner's data came back similarly. Thus it was decided that a surrogate target, YopH, would be pursued. Expression was started with the surrogate.
Image 8: Nanodrop of mini-prep on sample 2B from the liquid cultures that were grown from the re-done master plates showing a yield too low to submit to sequencing.
Image 7: Nanodrop of mini-prep on sample 1B from the liquid cultures that were grown from the re-done master plates showing a yield too low to submit to sequencing.
Image 6: Nanodrop of mini-prep on sample 11A from the liquid cultures that were grown from the re-done master plates showing a yield too low to submit to sequencing.
Image 5: Nanodrop of mini-prep on sample 10A from the liquid cultures that were grown from the re-done master plates showing a good yield high enough to send to sequencing.
Image 4: Nanodrop of mini-prep on sample 8A from the liquid cultures that were grown from the re-done master plates showing a very high yield.
Image 3: Nanodrop of mini-prep on sample 7A from the liquid cultures that were grown from the re-done master plates showing a yield just high enough to send to sequencing.
Image 2: Nanodrop of mini-prep on sample 4A from the liquid cultures that were grown from the re-done master plates showing a yield too low to submit to sequencing.
Image 1: Nanodrop of mini-prep on sample 1A from the liquid cultures that were grown from the re-done master plates showing a yield too low to submit to sequencing.
Good. Include pics of your nanodrops after mini prep -UM
Week 12
After the second attempt at cloning failed, the first master plate was returned to, and more attempts to grow liquid cultures were made. First, a fresh batch of LB and agar plates were made from scratch, and immediately used, with the contents double checked to ensure that the plates and LB being used were not causing the cultures lack of growth. Then, fresh cultures from the original plates, labeled as "B" were chosen and made a master plate with, and the cultures from the first master plate were re-plated and labeled "A". When all plated cultures grew, but the liquid cultures did not, the liquid cultures were grown again at room temperature. The liquid cultures that grew at room temperature were successful, and mini-prep was performed.
Image 4: Master plate of colonies 1-8B, showing that the colony growth is not due to a fault plate.
Image 3: Re-plated master plate of colonies 1-12A, showing that the colony growth is not due to a fault plate.
All 12 "A" colonies, and 8 "B" colonies were used to make fresh master plates, pictured above, and were put into liquid broth in order to be grown. All 20 colonies failed in the 37 degree incubator during the first round. Then eight colonies, (1A, 4A, 7A, 10A, 8A, 11A, 1B and 2B) were selected from the new master plates and grown at room temperature for 24 hours, and did grow successfully.
Image 2: Second original plate made, with numbers in black representing the "A" plates, the original master plate and the re-plated version, and blue numbers representing the first four of the eight colonies that were used to create a second master plate, labeled "B".
Image 1: First original plate made, with numbers in black representing the "A" plates, the original master plate and the re-plated version, and blue numbers representing the second four of the eight colonies that were used to create a second master plate, labeled "B".
Week 11
For this week, cloning was continually attempted with various components changed in order to attempt to grow a viable set of cultures.
The second attempt at cloning did not produce an original plate as the cultures did not grow.
Image 3: Figure 5: Gel run on preparation of pNIC after the first step of cloning protocol, the first lane shows a 1kb DNA ladder and the second shows the sample of the prepped pNIC. A reference ladder is provided on the left, showing that pNIC has been cut in the proper places.
Image 2: Figure 4: Screenshot of nanodrop performed on the second round of cloning prep of pNIC. In this round of cloning, the yield is almost double the yield from this point In the first round of cloning.
Notes:
The liquid cultures are not pictured because they did not show any growth. It was determined after that step of cloning was completed the first time that too much Kanamycin had been added, and that had probably killed off the cultures. Using the master plate, twelve new LB tubs were incubated with the cultures, however none of these grew either. This step was repeated a third time, this time warming the LB, but bacterial growth failed once again. A second round of cloning was started in the meantime to try to prevent anymore lost time.
Image 1: Figure 3: Master plate made from first attempt at cloning with pNIC and added Vibrio cholerae Phosphoserine phosphatase. Twelve regions were created on the master plate, and six colonies from each of the two original plates were selected to be put unto the master plate. As can be seen, all twelve of the cultures on the master plate grew, however the corresponding liquid cultures did not.
Where is Week 10? Missing plate pic. -UM
Week 9 and 10
For weeks 9 and 10, the first round of cloning was performed. To minimize chance for error, the protocol was followed on back to back days until the end of the master plate protocol. However, it was found at the very end of this step that the tubes from the master plate protocol had received too much Kanamycin and will have to be redone.
There is not yet a picture of the master plate, but it should be noted that while the colonies in the tubes did not grow, there was growth in all twelve sectors of the master plate.
Figure 2: Gel run on preparation of pNIC after the first step of cloning protocol; while the lanes seem to have been damaged, the first lane shows the sample of the prepped pNIC, while the second shows a 1kb DNA ladder. Compared to the image of the ladder provided, it appears as though it has been cut in the proper places.
Figure 1: Screenshot of the nanodrop performed after the preparation of pNIC from the cloning protocol, showing a decent yield.
Week 8
For week 8, midiprep and the transformation of pNIC were done. The first transformation was completed at the end of week 7, and then the first midiprep, second transformation of pNIC and second midiprep were all completed within week 8.
The second attempt at midiprep proved even worse for yield, and was not fit to move on into cloning. Because of time, at this point midiprep was abandoned and another sample from a midiprep performed by Will E. was used for cloning in week 9.
Image 5: Screenshot of second nanodrop done on second attempt at midiprep, showing very low yield.
Image 4: Screenshot of nanodrop done on second attempt at midiprep, showing very low yield.
With the low yield produced by midiprep, it was decided that transformation and midiprep should be repeated for a better yield.
Image 3: Screenshot of second nanodrop done on first attempt at midiprep, showing very low yield.
Image 2: Screenshot of nanodrop done on first attempt at midiprep, showing very low yield.
Image 1: Picture of the transformation of pNIC (at the end in pellet form), from the end of week 7.
Very good! Have you tried Gel extraction to get rid of that contam. from pcr squared? -UM
Week 7
Work on PCR was continued throughout this week, and transformation of PNIC was started.
The fourth round of squared was cleaned up and nanodropped, and showed a high yield.
Figure 5: Image of the nanodrop done of the fourth round of PCR squared, showing a ng/uL yield of 157.5, indicating that PCR has worked.
The fourth round of squared was finally successful.
Figure 4:Gel showing fourth attempt at PCR squared with a 1kb ladder; this one is suspected to be successful, and will move on to PCR clean-up. Lane 1: 1kb ladder Lane 2: sample 1 of PCR squared Lane 3: sample 2 of PCR squared Lane 4: sample 3 of PCR squared Lane 5: sample 4 of PCR squared
The third attempt at squared failed because of a gel error.
Figure 3: Gel showing third attempt at PCR squared; this failure is suspected to be caused by having been run in a mislabeled rig that most likely contained TBE, not also that there are only three samples because one sample was spilt. Lane 1: 1kb ladder Lane 2: sample 1 of PCR squared Lane 3: sample 2 of PCR squared Lane 4: sample 3 of PCR squared
The first two rounds of squared were not considered successful because the bands were not bright enough.
Figure 2: Gel showing first and second attempts at PCR squared with 1kb ladder, the lightness of the bands and the smearing indicates that it must be redone. Lane 1: blank Lane 2: 1 kb ladder Lane 3: 2nd attempt at squared, sample 1 Lane 4: 2nd attempt at squared, sample 2 Lane 5: 2nd attempt at squared, sample 3 Lane 6: 2nd attempt at squared, sample 4 Lane 7: 1st attempt at squared, sample 1 Lane 8: 1st attempt at squared, sample 2 Lane 9: 1st attempt at squared, sample 3 Lane 10: 1st attempt at squared, sample 4
The fourth attempt at secondary was finally successful.
Figure 1: Gel showing fourth attempt at secondary against a 1 kb ladder; this round was considered a success. Lane 1: secondary PCR Lane 2: 1 kb ladder
Good images and captions. Very good analysis! Now just cut back on the human errors and hopefully things will work better! - Michael T
Week 5 & 6
PCR for the Target Vibrio Cholerae was run throughout the two weeks. During this time period, dilutions, LB, and LB agar plates were also made for future use.
Third attempt at Secondary:
Secondary was attempted again with the same primary sample. For this round of secondary, the temperature during the annealing step was raised by two degrees, as suggested by William (also working on the target) who used those qualifications to get his secondary to work. The times were also adjusted to pick the middles times for each of the steps, to shorten the steps in order to try to get secondary to work. Ultimately, the times were probably shortened too much, because nothing showed up at all on this gel run for this attempt at PCR. It is also suspected that there was a problem with the gel because of the distortion as seen in the image. It should be noted that while the lane that was supposed to contain secondary did not show up on the image, when looking at the gel directly, a blue marker could be seen in the second lane.
Figure 2: Gel image with ladder of secondary PCR redone with changes. Lane one contains the 1kb ladder, and lane two had a sample of the secondary PCR, which does not show up on this image.
Second attempt at Primary and Secondary PCR:
Primary and secondary were both run before putting on a gel a second time, this time, it was noted that the wrong times had been used in the first attempt, and the correct times were used. Primary PCR left a smear and worked, but secondary smeared as well, indicating it did not work.
Image 1: Gel image of the second attempt at primary and secondary PCR, with a 1 kb ladder in the first lane, primary in the second lane, and secondary in the third lane. The second lane showing primary shows an appropriate smear, indicating primary worked, while the third lane with secondary shows a smear as well, indicating it did not work.
First attempt at Primary and Secondary PCR:
Primary and secondary were both run before putting on a gel. Due to pipette error, all of each of the samples was taken out to mix with blue dye for a run on the gel. Once the gel was run, it was discovered that someone had mixed in ladder to the blue dye, and so all of the primary and secondary samples were contaminated, and had to be redone.
Week 3 & 4
Dax - crop your image in the imaging software, include a ladder image. Missing a Primary PCR run. Otherwise - looks good. - Dr. B 092713
Week 4
Second PCR of pGbr
Figure 1: Second PCR of pGbr 22, with lane 1 containing 100 bp ladder, lane 2 containing sample D, lane 3 containing sample B, lane 4 containing sample C, and lane 5 containing sample A.
Note that sample D was accidentally placed in lane 2, and sample A was placed in lane 5.
Analysis:
This is a successful PCR of pGbr 22, showing nothing in lane 2, where sample D, without DNA is located, and gaining brightness going from sample A to C.
Week 3
First PCR of pGbr
Figure 1: Gel from the first PCR of pGbr22 showing 100 bp ladder in lane 1, and samples A-D in order, in lanes 2-5 respectively.
Analysis: The first PCR pf pGbr22 failed, with sample D being the only lane that showed, and sample D supposedly being the sample not containing DNA. It is suspected that cross contamination is the cause.
RE Digest of pGbr 22
Figure 1: RE Digest of pBbr22 in lanes 6-10, with lane 6 containing a 1kb ladder, lane 7 containing uncut plasmid, lane 8 the sample with EcoRI, lane 9 sample with PvuII, and lane 10 containing the sample with EcoRI and PvuII together.
Analysis: Compared with the ladder, we can see only a smear with the uncut plasmid, and different markers for where each of the following samples cut the DNA sequence, with the sections being compiled in the last lane that contains both EcoRI and PvuII.
Primer Tail Design:
Figure 1: PNIC as cut by Bsa1
Figure 2: The gel of PNIC as cut by Bsa1 with 1000kb ladder shown and both cut sites shown, with coordinates and length listed in key on right.
Figure 3: Our to-be-inserted sequence, showing a Bsa1 cut site already present prior to insertion.
Figure 4: PNIC with our insert showing the Bsa1 cut site that was present in the sequence, prior to insertion
Figure 5: The gel of PNIC with our insert with 1000kb ladder shown and the site and length of the cut shown in the key on the right.
Phosphoserine Phosphotase Vibrio Cholerae Primer Tale Design
Sequence to be inserted into PNIC, with the start and end highlighted in green, and the tails highlighted in yellow.
Note that base pairs were added and removed from both ends until the 2mM of Mg2+ had temperatures of 72.4° and 69.8° Celsius for the forward and reverse primers respectively.
Primers to Order:
Upstream:
TACTTCCAATCCATGGACGCGCTGACCA
Downstream (reverse complemented):
TATCCACCTTTACTGTTACGGTTTAGATTTCCAAGAGAG
*Note that this insert contains code that codes for a cut by BsaI.
Full pNIC-Bsa4 sequence with insert, start and end highlighted in green, and the 6-His tag highlighted in yellow.
Week 1 & 2
Dax - good job. Need your PCR results Include a one sentence analysis for each exepriment. Dr. B 090913
Analyzing DNA:
Figure 1: Comparison between sequence and human; math is poor with only 3% query coverage.
Figure 2: Comparison with nucleotides; the top two matches are strong, with 60% query coverage and a 100% identity match with montipora efflorescens
Figure 3: Comparison between PurpleProtein file and given, showing 100% query coverage, and 99% identity match.
Figure 4: Output from the ORF finder showing six frames
Figure 5: Comparison of PurpleProtein translated sequence, and original translated sequence in BLAST showing 99% query coverage and a 100% identity match.
Image 6: Predicted gel images of EcoRI, PvuII, and both together in order from left to right with the lanes labeled "marker" indicated the gel of the ladder 1kb for reference.
Oligo Design:
Figure 1: Oligo primer sequence for target phosphoserine phosphatase (Vibrio cholerae) as submitted with codon optimization for E.Coli.
Week 1
Nanodrop:
Figure 1: Absorbance curve of pGBR 22 read with concentration 198.1 ng/uL
Figure 2: Absorbance curve of pGBR22 read with concentration 195.3 ng/uL
The known concentration was listed at 205.4 ng/uL.
The average concentration found was 196.7 ng/uL.
Note that the measurements were taken several more times than listed to ensure the measurement was correct.
Enzyme assay and inhibition assays were done this week. Virtual was attempted, but due to fault in preparation of files, was not able to run on the processors and yield any results.
Week 14
On this week, expression, purification, FPLC, characterization, and storage were completed.
Week 13
On this week, mini-prep and submitting to DNA sequencing were performed. The DNA sequencing results came back blank or with very, very few codons. My partner's data came back similarly. Thus it was decided that a surrogate target, YopH, would be pursued. Expression was started with the surrogate.
Good. Include pics of your nanodrops after mini prep -UM
Week 12
After the second attempt at cloning failed, the first master plate was returned to, and more attempts to grow liquid cultures were made. First, a fresh batch of LB and agar plates were made from scratch, and immediately used, with the contents double checked to ensure that the plates and LB being used were not causing the cultures lack of growth. Then, fresh cultures from the original plates, labeled as "B" were chosen and made a master plate with, and the cultures from the first master plate were re-plated and labeled "A". When all plated cultures grew, but the liquid cultures did not, the liquid cultures were grown again at room temperature. The liquid cultures that grew at room temperature were successful, and mini-prep was performed.
All 12 "A" colonies, and 8 "B" colonies were used to make fresh master plates, pictured above, and were put into liquid broth in order to be grown. All 20 colonies failed in the 37 degree incubator during the first round. Then eight colonies, (1A, 4A, 7A, 10A, 8A, 11A, 1B and 2B) were selected from the new master plates and grown at room temperature for 24 hours, and did grow successfully.
Week 11
For this week, cloning was continually attempted with various components changed in order to attempt to grow a viable set of cultures.
The second attempt at cloning did not produce an original plate as the cultures did not grow.
Notes:
The liquid cultures are not pictured because they did not show any growth. It was determined after that step of cloning was completed the first time that too much Kanamycin had been added, and that had probably killed off the cultures. Using the master plate, twelve new LB tubs were incubated with the cultures, however none of these grew either. This step was repeated a third time, this time warming the LB, but bacterial growth failed once again. A second round of cloning was started in the meantime to try to prevent anymore lost time.
Where is Week 10? Missing plate pic. -UM
Week 9 and 10
For weeks 9 and 10, the first round of cloning was performed. To minimize chance for error, the protocol was followed on back to back days until the end of the master plate protocol. However, it was found at the very end of this step that the tubes from the master plate protocol had received too much Kanamycin and will have to be redone.
There is not yet a picture of the master plate, but it should be noted that while the colonies in the tubes did not grow, there was growth in all twelve sectors of the master plate.
Week 8
For week 8, midiprep and the transformation of pNIC were done. The first transformation was completed at the end of week 7, and then the first midiprep, second transformation of pNIC and second midiprep were all completed within week 8.
The second attempt at midiprep proved even worse for yield, and was not fit to move on into cloning. Because of time, at this point midiprep was abandoned and another sample from a midiprep performed by Will E. was used for cloning in week 9.
With the low yield produced by midiprep, it was decided that transformation and midiprep should be repeated for a better yield.
Very good! Have you tried Gel extraction to get rid of that contam. from pcr squared? -UM
Week 7
Work on PCR was continued throughout this week, and transformation of PNIC was started.
The fourth round of squared was cleaned up and nanodropped, and showed a high yield.
The fourth round of squared was finally successful.
The third attempt at squared failed because of a gel error.
The first two rounds of squared were not considered successful because the bands were not bright enough.
The fourth attempt at secondary was finally successful.
Good images and captions. Very good analysis! Now just cut back on the human errors and hopefully things will work better! - Michael T
Week 5 & 6
PCR for the Target Vibrio Cholerae was run throughout the two weeks. During this time period, dilutions, LB, and LB agar plates were also made for future use.
Third attempt at Secondary:
Secondary was attempted again with the same primary sample. For this round of secondary, the temperature during the annealing step was raised by two degrees, as suggested by William (also working on the target) who used those qualifications to get his secondary to work. The times were also adjusted to pick the middles times for each of the steps, to shorten the steps in order to try to get secondary to work. Ultimately, the times were probably shortened too much, because nothing showed up at all on this gel run for this attempt at PCR. It is also suspected that there was a problem with the gel because of the distortion as seen in the image. It should be noted that while the lane that was supposed to contain secondary did not show up on the image, when looking at the gel directly, a blue marker could be seen in the second lane.
Second attempt at Primary and Secondary PCR:
Primary and secondary were both run before putting on a gel a second time, this time, it was noted that the wrong times had been used in the first attempt, and the correct times were used. Primary PCR left a smear and worked, but secondary smeared as well, indicating it did not work.
First attempt at Primary and Secondary PCR:
Primary and secondary were both run before putting on a gel. Due to pipette error, all of each of the samples was taken out to mix with blue dye for a run on the gel. Once the gel was run, it was discovered that someone had mixed in ladder to the blue dye, and so all of the primary and secondary samples were contaminated, and had to be redone.
Week 3 & 4
Dax - crop your image in the imaging software, include a ladder image. Missing a Primary PCR run. Otherwise - looks good. - Dr. B 092713
Week 4
Second PCR of pGbr
Note that sample D was accidentally placed in lane 2, and sample A was placed in lane 5.
Analysis:
This is a successful PCR of pGbr 22, showing nothing in lane 2, where sample D, without DNA is located, and gaining brightness going from sample A to C.
Week 3
First PCR of pGbr
Analysis: The first PCR pf pGbr22 failed, with sample D being the only lane that showed, and sample D supposedly being the sample not containing DNA. It is suspected that cross contamination is the cause.
RE Digest of pGbr 22
Analysis: Compared with the ladder, we can see only a smear with the uncut plasmid, and different markers for where each of the following samples cut the DNA sequence, with the sections being compiled in the last lane that contains both EcoRI and PvuII.
Primer Tail Design:
Phosphoserine Phosphotase Vibrio Cholerae Primer Tale Design
Sequence to be inserted into PNIC, with the start and end highlighted in green, and the tails highlighted in yellow.
TACTTCCAATCCATGGACGCGCTGACCACCCTCCCGATCAAAAAGCACACCGCGCTGCTGAACCGTTTC CCGGAAACCCGCTTCGTTACCCAACTGGCGAAAAAGCGTGCGTCTTGGATCGTTTTCGGTCAC
TACCTCACTCCAGCACAGTTTGAAGATATGGATTTTTTCACCAATCGTTTCAATGCGATC
CTGGACATGTGGAAAGTTGGCCGTTACGAAGTTGCGCTGATGGACGGTGAACTGACCTCT
GAACACGAAACCATCCTGAAAGCGCTGGAACTCGACTACGCTCGCATCCAGGACGTTCCA
GACCTCACCAAACCGGGCCTGATCGTTCTCGACATGGACTCTACCGCTATCCAGATCGAA
TGCATCGACGAAATTGCGAAGCTGGCGGGTGTTGGCGAGGAAGTGGCCGAAGTTACGGAA
CGTGCGATGCAGGGCGAGCTGGACTTCGAACAGTCTCTGCGTCTGCGTGTTTCTAAACTC
AAAGACGCCCCTGAACAGATCCTGAGCCAGGTTCGTGAAACGCTGCCGCTCATGCCTGAA
CTGCCGGAACTGGTTGCGACCCTGCACGCGTTCGGTTGGAAGGTAGCAATCGCGTCTGGT
GGTTTCACCTACTTTTCTGACTACCTGAAGGAACAACTCAGCCTCGATTACGCGCAGTCT
AACACCCTGGAAATTGTTTCTGGTAAACTGACTGGTCAAGTTCTGGGTGAAGTTGTGTCT
GCTCAGACCAAAGCGGACATCCTGCTGACCCTGGCGCAACAGTACGACGTTGAAATCCAC
AACACCGTTGCGGTGGGTGACGGTGCGAACGACCTGGTTATGATGGCGGCTGCGGGCCTC
GGTGTAGCGTACCATGCGAAACCGAAGGTTGAGGCGAAGGCGCAGACCGCAGTTCGTTTC
GCTGGTCTCGGTGGTGTCGTTTGCATCCTGTCTGCGGCGCTCGTTGCGCAGCAAAAACTC
TCTTGGAAATCTAAACCGTAACAGTAAAGGTGGATA
Forward Primer: 30 bp GC content: 56.7%
Reverse Primer: 33 bp GC content: 36.4%
Note that base pairs were added and removed from both ends until the 2mM of Mg2+ had temperatures of 72.4° and 69.8° Celsius for the forward and reverse primers respectively.
Primers to Order:
Upstream:
TACTTCCAATCCATGGACGCGCTGACCA
Downstream (reverse complemented):
TATCCACCTTTACTGTTACGGTTTAGATTTCCAAGAGAG
*Note that this insert contains code that codes for a cut by BsaI.
Full pNIC-Bsa4 sequence with insert, start and end highlighted in green, and the 6-His tag highlighted in yellow.
TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCACCATCATCATCATCAT
TCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGGACGCGCTGACCACCCTCCCGATCAAAAAGCACACCGCGCTGCTGAACCGTT
TCCCGGAAACCCGCTTCGTTACCCAACTGGCGAAAAAGCGTGCGTCTTGGATCGTTTTCGGTCACTACCTCACTCCAGCACAGTTTGAAGATATGGA
TTTTTTCACCAATCGTTTCAATGCGATCCTGGACATGTGGAAAGTTGGCCGTTACGAAGTTGCGCTGATGGACGGTGAACTGACCTCTGAACACGAA
ACCATCCTGAAAGCGCTGGAACTCGACTACGCTCGCATCCAGGACGTTCCAGACCTCACCAAACCGGGCCTGATCGTTCTCGACATGGACTCTACCG
CTATCCAGATCGAATGCATCGACGAAATTGCGAAGCTGGCGGGTGTTGGCGAGGAAGTGGCCGAAGTTACGGAACGTGCGATGCAGGGCGAGCTGGA
CTTCGAACAGTCTCTGCGTCTGCGTGTTTCTAAACTCAAAGACGCCCCTGAACAGATCCTGAGCCAGGTTCGTGAAACGCTGCCGCTCATGCCTGAA
CTGCCGGAACTGGTTGCGACCCTGCACGCGTTCGGTTGGAAGGTAGCAATCGCGTCTGGTGGTTTCACCTACTTTTCTGACTACCTGAAGGAACAAC
TCAGCCTCGATTACGCGCAGTCTAACACCCTGGAAATTGTTTCTGGTAAACTGACTGGTCAAGTTCTGGGTGAAGTTGTGTCTGCTCAGACCAAAGC
GGACATCCTGCTGACCCTGGCGCAACAGTACGACGTTGAAATCCACAACACCGTTGCGGTGGGTGACGGTGCGAACGACCTGGTTATGATGGCGGCT
GCGGGCCTCGGTGTAGCGTACCATGCGAAACCGAAGGTTGAGGCGAAGGCGCAGACCGCAGTTCGTTTCGCTGGTCTCGGTGGTGTCGTTTGCATCC
TGTCTGCGGCGCTCGTTGCGCAGCAAAAACTCTCTTGGAAATCTAAACCGTAACAGTAAAGGTGGATACGGATCCGAATTCGAGCTCCGTCGACAAGCTTG
CGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCAT
AACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCG
GCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCC
GTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGC
CCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGAT
TTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCA
CTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGA
AACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCT
GGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATC
CGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTG
ATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATT
TTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCG
GAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTT
CCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAG
ACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGA
GCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGT
TTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCA
AGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTA
CCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAG
AAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGG
TATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTT
ACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCA
GCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGC
ACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACG
CGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAG
GCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCT
GATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGA
TGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGG
GTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGT
TTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCAT
TCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTC
GCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGC
GGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCG
GAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTC
GGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAAC
AGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATAT
AACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGG
CAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGAT
TGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTC
CACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAG
CAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCAT
CGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAAC
GACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCAC
CACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCA
TACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCG
CCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCG
AGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGAT
CCCGCGAAAT
Week 1 & 2
Dax - good job. Need your PCR results Include a one sentence analysis for each exepriment. Dr. B 090913
Analyzing DNA:
Oligo Design:
Week 1
Nanodrop:
The known concentration was listed at 205.4 ng/uL.
The average concentration found was 196.7 ng/uL.
Note that the measurements were taken several more times than listed to ensure the measurement was correct.